Proteins which inhibit protein synthesis have been isolated from various organisms including plants, bacteria and fungi. These protein toxins are thought to be produced by the organisms in order to provide a selective advantage for the growth of the organisms that produce them. Despite the divergent evolutionary background of the organisms in which these protein toxins are found, most toxins have strikingly similar mechanisms of action. One particular group of toxins exerts its action by blocking protein synthesis either by directly modifying elongation factor 2 (EF-2) or by modifying the ribosome itself so that EF-2 cannot function in protein synthesis. This class of toxins, ribosome-inactivating proteins (RIPs), can be isolated from plants of several families.
Plant ribosome-inactivating proteins have been divided into two groups based on their structure. Type I ribosome-inactivating proteins (type I RIPs) contain a single chain that has ribosome-inactivating activity. Examples of type I RIPs include gelonin, saporin, trichosanthin and bryodin. Type II ribosome-inactivating proteins (type II RIPs) are comprised of two chains, an A chain that is able to inactivate EF-2, and a B chain, that contains a cell binding domain having lectin-like properties. The binding domain enables type II RIPs to bind many cell types and to kill those cells. Examples of type II RIPs are ricin and abrin.
Although the two types of ribosome-inactivating proteins differ in their structures, both types inhibit protein synthesis by inactivating the 60S subunit of eukaryotic ribosomes through cleavage of the N-glycosidic bond of the adenine residue at position 4324 of 28 S rRNA (Endo and Tsurugi 1987, J. Biol. Chem. 262:8128-8130; Stirpe, F. et al. 1988, Nucl. Acid Res.,16:1349-1357).
Ribosome-inactivating proteins have been isolated from several families of plants including the Cariophyllaceae, Cucurbitaceae, Euphorbiaceae and Phytolaccaceae. The toxins have been isolated particularly from the root, seeds and leaves of the plants. Comparisons have been made of the N-terminal amino acid sequences of RIPs isolated from the seeds of Gelonium multifiorum (Euphorbiaceae), Momordica charantia (Cucurbitaceae), Bryonia dioica (Cucurbitaceae), Saponaria officinalis (saporin-5a, saporin-5b, saporin-6a, saporin-6b) (Cariophyllaceae) and from the leaves of Saponaria officinalis (saporin-1). Complete amino acid sequences have been determined for a Type I RIP from Trichosanthes kirilowii maxim and from Barley seed protein synthesis inhibitor. These comparisons show that at least the N-terminal regions of the toxins bryodin and momordin (members of the Curcurbitaceae family) show a high level of similarity with ricin A chain and with gelonin which are members of the Euphorbiaceae family. The similarity is thought to be a consequence of a similar evolutionary origin. Very little similarity was found between RIPs of the Cucurbitaceae and Euphorbiaceae families and those of the Phytolaccaceae or Cariophyllaceae families (Montecucchi et al., 1989, Int. J. Peptide Protein Res. 33:263-267). Although similarities are found in the amino acid sequences of the N-terminal regions of RIPs isolated from the same species, many differences do exist particularly between toxins isolated from different tissue of the same plant.
A plant protein toxin designated bryodin was initially identified as a 27-30 kDal protein isolated from the root of Bryonia dioica (United Kingdom Patent Application GB2194948, published Mar. 23, 1988). The toxin is a type I ribosome-inactivating protein having a single chain and a mechanism of action. which inactivates ribosomes by blocking productive interactions with elongation factor-2. In not having a cell binding domain, bryodin, like the other type I RIPs, does not normally bind to mammalian cells. The protein has been shown to have a molecular weight by gel filtration of about 27,300 daltons and about 28,800 daltons by polyacrylamide gel electrophoresis, and an isoelectric point of 9.5. This toxin was found to inhibit protein synthesis in the rabbit reticulocyte lysate system with wheat germ ribosomes at 3.6 ng/ml (ID.sub.50) and an LD.sub.50 in mice of 14.5 mg/kg when administered intraperitoneally. The N-terminal amino acid sequence has been determined to be ##STR1##
A second ribosome-inactivating protein has been isolated from the leaves of B. dioica (European Patent Publication EPO 390 040, published Oct. 3, 1990). This molecule has been described as having a molecular weight of 27,300 daltons by gel filtration and 28,800 daltons by polyacrylamide gel electrophoresis, and an isoelectric point of 9.5 and has been designated bryodin-L. This form of bryodin was found to inhibit protein synthesis in a rabbit reticulocyte lysate system with an EC.sub.50 of 0.1 nM (3.6 ng/ml) and has an LD.sub.50 in mice of 10 mg/kg when administered intraperitoneally. An amino acid analysis was also provided, but no amino acid sequence has been disclosed.
Ribosome-inactivating proteins are of interest because of their usefulness as components of "immunotoxins." Immunotoxins are hybrid molecules consisting of a toxic moiety linked to an antibody capable of selectively directing the toxin to a specific target cell. Potential target cells include harmful cells, i.e. , neoplastic, virally infected, immunocompetent or parasitic cells. Immunotoxins as defined in the present invention can be chemical conjugates of a cell-specific ligand linked to a toxic molecule, such as a ribosome-inactivating protein. The fact that many different ribosome-inactivating proteins are known and that new toxins are being discovered provides a variety of toxic moieties which have varying levels of intrinsic toxicity on whole cells when unconjugated and provide an available source of alternative toxins should the patient develop an immune response during long term in vivo treatment to the originally administered immunotoxin. In addition, some immunotoxins, saporin 6 and an anti-Thy 1.1 antibody or its F(ab').sub.2 fragment, were more toxic than free toxin providing a need for new and different toxin molecules.
The present invention provides a novel plant protein toxin isolated from Bryonia dioica we have designated bryodin 2, which is distinguishable from bryodin and bryodin-L by its oligonucleotide sequence, amino acid sequence, amino acid composition, toxicity in animals and immunogenicity. Bryodin 2 provides a new ribosome-inactivating protein that can be used to form additional and possibly better immunotoxins and toxin fusion molecules for use in formulating pharmaceutical compositions for use in treating cancer, certain viral infections, modulating the immune response, and other diseases.